Method for the High Throughput Screening of Transposon Tagging Populations and Massive Parallel Sequence Identification of Insertion Sites

ABSTRACT

Method for the identification of a gene in a transposon population, comprising isolating genomic DNA, optionally pooling the DNA, restrict the DNA in the pools using an enzyme, ligate adaptors, amplifying the adaptor-ligated fragments with primers, one of which is a primer complementary to a border of a transposon sequence, high throughput sequencing of the fragments, aligning the fragments with known sequences in a database and thereby identifying gene candidates.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology andgenetics. The invention relates to improved strategies for identifyingmutants for genes in populations, based on the use of high throughputsequencing technologies

BACKGROUND OF THE INVENTION

Transposon tagging populations are used in modern plant genomicsresearch to identify genes affecting traits of agronomic or generalimportance by reverse genetics approaches.

They represent complementary tools for gene discovery, as transposonpopulations are commonly used to identify the gene responsible for anobserved phenotype, the so-called forward genetics approach. This isdistinguished in the art from the reverse genetics approach wherein,mutational events are identified in sequences (genes) of interest. Therate-limiting step for the methods is the screening work associated withidentification of the individual carrying a mutation in the gene orsequence of interest. Below, the principles of transposon populationsand the screening methods are described in more detail and moreefficient screening methods are presented which increase the value ofthese tools for gene-discovery.

Transposons are mobile genetic elements occurring, naturally orengineered, at multiple copies in the genome. They are unstable as theirposition in the genome can change by excision and insertion at novelsites, usually at any given moment in the life cycle. Transposonpopulations are valuable for gene-discovery because they can disruptgene function if they insert in gene sequences or their regulatoryregions. The sequences of many transposons used in plant breeding areknown, but once a plant with an interesting phenotype is observed, it isnot known which gene is affected by transposon insertion. It is, ingeneral, also not known if and if so, which, transposon is responsiblefor the phenotype. Depending on the organism and transposon, copynumbers of transposons in transposon populations range from several tensto hundreds of transposon per plant.

Current screening methods for analysis of transposon-induced phenotypicmutant sequences include linked-PCR based methods in order to obtainflanking sequences from sequence-specific transposon integration sites.A limitation of linker-PCR is that determination of flanking sequencesrequires band-excision from sequencing gels, which is time-consuming,difficult to automate and relatively low-throughput (not easilyadaptable to thousands of bands).

Screening transposon populations would be improved if a simple methodwould be available to collect flanking sequences of all or at least partof the transposons, integrated in the genome. Here we seek to provide anefficient approach to analyse and use insertion events in preferredsequences.

DEFINITIONS

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference. Transposon: Transposons are sequences ofDNA that can move around to different positions within the genome of asingle cell, a process called Transposition. In the process, they cancause mutations and change the amount of DNA in the genome. Transposonsare also called “jumping genes” or “mobile genetic elements”. There area variety of mobile genetic elements; they can be grouped based on theirmechanism of transposition. Class I mobile genetic elements, orretrotransposons, move in the genome by being transcribed to RNA andthen back to DNA by reverse transcriptase, while class II mobile geneticelements move directly from one position to another within the genomeusing a transposase to “cut and paste” them within the genome.Transposition can be replicative wherein one copy of the transposableelement remains at the donor site and another is inserted at the targetsite; or transposition can occur conservatively wherein the transposableelement is excised from one site and inserted at the other. The termincludes, but is not limited to, transposable elements found inprokaryotes such as insertion sequences (IS), transposons (Tn), orbacteriophages such as Mu and D108. Eukaryotic transposable elementsinclude, but are not limited to: Copia elements as are found in D.melanogaster; TY elements such as those found in yeast; Ta1 and Tnt 1transposable elements such as those found in Arabidopsis; IAP found inmice; Tam or Cin transposable elements such as those found insnapdragon; and AC, Spm, Bs, Cin, Dt, and Mutator transposable elementssuch as those found in maize. The term is also inclusive of synthetictransposable elements which can insert themselves either replicativelyor conservatively within a host genome and whose transposition orexcision from the genome can be controlled by human intervention. Forexample, a synthetic transposable element can be constructed which lacksa functional transposase (the enzyme that mediates transposition) butwhich is supplied in trans by operably linking the transposase gene toan inducible promoter. Transposon population: A population of individualfrom one organism (usually plants, but other organisms, such asDrosophila and mouse are also possible), each of which carrying aplurality of transposons in their genome and each of which transposonsmay affect one or more genes, resulting in different phenotypes.Typically transposon populations can be obtained selected fromindividuals or varieties that express instability in a phenotypic trait.Transposon populations may vary widely in size, and for certainpurposes, partial populations can be used that contain 90, 80 70, 60,50, 40 30 or even only 20% of the original population.

Tag: A short sequence that can be added to a primer or included in itssequence or otherwise used as label to provide a unique identifier. Sucha sequence identifier can be a unique base sequence of varying butdefined length uniquely used for identifying a specific nucleic acidsample. For instance 4 bp tags allow 4(exp4)=256 different tags. Typicalexamples are ZIP sequences, known in the art (Iannone et al. Cytometry39:131-140, 2000). Using such a tag, the origin of a PCR sample can bedetermined upon further processing. In the case of combining processedproducts originating from different nucleic acid samples, the differentnucleic acid samples are generally identified using different tags. Inthe case of the present invention, the addition of a unique sequence tagserves to identify the co-ordinates of the individual plant in the poolof sequences amplification products. Multiple tags can be used.

Tagging: refers to the process of the addition of a tag or label to anucleic acid in order to be able to distinguish it from a second orfurther nucleic acid. Tagging can be performed, for example, by theaddition of a sequence identifier during amplification by using taggedprimers or by any other means known in the art.

Restriction endonuclease: a restriction endonuclease or restrictionenzyme is an enzyme that recognises a specific nucleotide sequence(target site) in a double-stranded DNA molecule, and will cleave bothstrands of the DNA molecule at every target site.

Restriction fragments: the DNA molecules produced by digestion with arestriction endonuclease are referred to as restriction fragments. Anygiven genome (or nucleic acid, regardless of its origin) will bedigested by a particular restriction endonuclease into a discrete set ofrestriction fragments. The DNA fragments that result from restrictionendonuclease cleavage can be further used in a variety of techniques andcan for instance be detected by gel electrophoresis.

Ligation: the enzymatic reaction catalyzed by a ligase enzyme in whichtwo double-stranded DNA molecules are covalently joined together isreferred to as ligation. In general, both DNA strands are covalentlyjoined together, but it is also possible to prevent the ligation of oneof the two strands through chemical or enzymatic modification of one ofthe ends of the strands. In that case the covalent joining will occur inonly one of the two DNA strands.

Synthetic oligonucleotide: single-stranded DNA molecules havingpreferably from about 10 to about 50 bases, which can be synthesizedchemically are referred to as synthetic oligonucleotides. In general,these synthetic DNA molecules are designed to have a unique or desirednucleotide sequence, although it is possible to synthesize families ofmolecules having related sequences and which have different nucleotidecompositions at specific positions within the nucleotide sequence. Theterm synthetic oligonucleotide will be used to refer to DNA moleculeshaving a designed or desired nucleotide sequence.

Adaptors: short double-stranded DNA molecules with a limited number ofbase pairs, e.g. about 10 to about 30 base pairs in length, which aredesigned such that they can be ligated to the ends of restrictionfragments. Adaptors are generally composed of two syntheticoligonucleotides which have nucleotide sequences which are partiallycomplementary to each other. When mixing the two syntheticoligonucleotides in solution under appropriate conditions, they willanneal to each other forming a double-stranded structure. Afterannealing, one end of the adaptor molecule is designed such that it iscompatible with the end of a restriction fragment and can be ligatedthereto; the other end of the adaptor can be designed so that it cannotbe ligated, but this needs not be the case (double ligated adaptors).

Adaptor-ligated restriction fragments: restriction fragments that havebeen capped by adaptors.

Nucleic acid: a nucleic acid according to the present invention mayinclude any polymer or oligomer of pyrimidine and purine bases,preferably cytosine, thymine, and uracil, and adenine and guanine,respectively (See Albert L. Lehninger, Principles of Biochemistry, at793-800 (Worth Pub. 1982) which is herein incorporated by reference inits entirety for all purposes). The present invention contemplates anydeoxyribonucleotide, ribonucleotide or peptide nucleic acid component,and any chemical variants thereof, such as methylated, hydroxymethylatedor glycosylated forms of these bases, and the like. The polymers oroligomers may be heterogenous or homogenous in composition, and may beisolated from naturally occurring sources or may be artificially orsynthetically produced. In addition, the nucleic acids may be DNA orRNA, or a mixture thereof, and may exist permanently or transitionallyin single-stranded or double-stranded form, including homoduplex,heteroduplex, and hybrid states.

Sequencing: The term sequencing refers to determining the order ofnucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA.

Aligning and alignment: With the term “aligning” and “alignment” ismeant the comparison of two or more nucleotide sequences based on thepresence of short or long stretches of identical or similar nucleotides.Several methods for alignment of nucleotide sequences are known in theart, as will be further explained below. Sometimes the terms “assembly”or “clustering” are used as synonyms.

High-throughput screening: High-throughput screening, often abbreviatedas HTS, is a method for scientific experimentation especially relevantto the fields of biology and chemistry. Through a combination of modernrobotics and other specialised laboratory hardware, it allows aresearcher to effectively screen large amounts of samplessimultaneously.

Primers: in general, the term primer refers to a DNA strand which canprime the synthesis of DNA. DNA polymerase cannot synthesise DNA de novowithout primers: it can only extend an existing DNA strand in a reactionin which the complementary strand is used as a template to direct theorder of nucleotides to be assembled. We will refer to the syntheticoligonucleotide molecules which are used in a polymerase chain reaction(PCR) as primers.

Primers with increased affinity: Primers containing modified nucleotidessuch as PNA or LNA, which increase their thermal stability, which allowsfor more specific amplification based on single nucleotide sequencedifferences. In order to achieve this, one or several modifiednucleotides are often included, preferably at the 3′ end of the primer.

DNA amplification: the term DNA amplification will be typically used todenote the in vitro synthesis of double-stranded DNA molecules usingPCR. It is noted that other amplification methods exist and they may beused in the present invention without departing from the gist thereof.

Selective hybridisation: relates to hybridisation, under stringenthybridisation conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence to a detectably greater degree (e.g.,preferably at least 2-fold over background) than its hybridisation tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. The terms “stringent conditions” or “stringenthybridisation conditions” includes reference to conditions under which aprobe will hybridise to its target sequence, to a detectably greaterdegree than other sequences (e.g., preferably at least 2-fold overbackground). Stringent conditions are sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridisation and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about100 nucleotides in length, preferably no more than 50, or 25 nucleotidesin length. Typically, stringent conditions will be those in which thesalt concentration is less than about 1.5 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at a pH of about 7.0 to8.3 and the temperature is typically at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and typically at least about 60° C.for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilisingagents such as formamide.

Exemplary low stringency conditions include hybridisation with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecylsulphate) at 37° C., and a wash in 1* to 2*SSC (20*SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridisation in 40 to 45% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.5* to 1*SSC at 55 to 60° C.Exemplary high stringency conditions include hybridisation in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1*SSC at 60 to65° C. Specificity is typically the function of post-hybridisationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the Tm can be approximatedfrom the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284(1984): Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; whereM is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridisation solution, and L is the length of thehybrid in base pairs. The Tm is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridises to a perfectly matched probe. Tm is reduced by about 1° C.for each 1% of mismatching; thus, Tm, hybridisation and/or washconditions can be adjusted to hybridise to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theTm can be decreased 10° C. Generally, stringent conditions are selectedto be about 5° C. lower than the thermal melting point (Tm) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilise a hybridisationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point(Tm); moderately stringent conditions can utilise a hybridisation and/orwash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm);low stringency conditions can utilise a hybridisation and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm).Using the equation, hybridisation and wash compositions, and desired Tm,those of ordinary skill will understand that variations in thestringency of hybridisation and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a Tm of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution) it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridisation of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridisation with Nucleic Acid Probes, Part 1, Chapter 2“Overview of principles of hybridisation and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

DESCRIPTION OF THE INVENTION

The present inventors have found that by using high throughputsequencing strategies, the above-mentioned goals can be achieved andtransposon populations, or populations comprising members carryingphenotypes of interest caused by transposon insertions, can beefficiently screened for the presence of insertions into genes ofinterest.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for the identification of an insertionassociated with a gene or sequence of interest in a member of atransposon population, comprising the steps of:

-   -   (a) isolating, individually or in pools, genomic DNA of the        transposon population;    -   (b) optionally, pooling the DNA obtained in step (a);    -   (c) restrict the DNA using one or more, preferably two or more,        most preferably two, restriction endonucleases, preferably at        least one of which is a frequent cutting restriction        endonuclease that does not cut in the transposon and preferably        at least one is a rare cutting restriction endonuclease that        cuts in the transposon, ligate adaptors to the restriction        fragments to thereby prepare adaptor-ligated restriction        fragments;    -   (d) amplifying the adaptor-ligated restriction fragments with a        pair of (optionally labelled) primers, whereby one of the        primers comprises a section that is complementary (capable of        hybridising) to part of a (known) transposon sequence and        further contains a sequence primer binding site, wherein the        other primer is at least complementary to the adaptor, wherein        one or both primers contain a tag;    -   (e) optionally, pooling the amplification products of step (d)        to create a library of amplification products;    -   (f) optionally, fragmenting the amplification products in the        library;    -   (g) determining the nucleotide sequence of the fragments of        (d), (e) or (f) using high throughput sequencing;    -   (h) optionally, trimming the sequence of the fragments in silico        to thereby remove any adaptor and/or transposon related sequence        information;    -   (i) identifying one or more fragments of step (g) or (h) that        are capable of aligning with nucleotide sequences from a        database, thereby correlating the nucleotide sequences from the        database with the phenotype of interest;    -   (j) identifying the member(s) of the transposon population        containing the fragment(s) of step (i);    -   (k) optionally, designing a probe or PCR primer pair based on        the fragments of step (i) and using it to confirm transposon        insertion in the gene of interest in the genome of the member        identified in (j).

The isolation of DNA to provide for DNA samples of each member in thepopulation is generally achieved using common methods in the art such asthe collection of tissue from a member of the population, DNA extraction(for instance using the Q-Biogene fast DNA kit), quantification andnormalisation to obtain equal amounts of DNA per sample. As an example,the present invention is illustrated based on a transposon population of1000 plants. Typically, DNA is isolated of each member of the populationexpressing the phenotype of interest.

In accordance with the method of the present invention, individualorganisms whose genomic DNA comprises at least one transposableelement-tagged gene can be segregated by the presence or absence of amutant phenotype of interest. Thus a method is provided that is suitablefor the identification and isolation of a genetic sequence from anorganism, wherein disruption of genomic DNA of said organism by atransposable element flanking said genetic sequence is associated,directly or indirectly, with a mutant phenotype

The mutant phenotype of the organism is preferably one known orsuspected of arising from disruption of a single gene by insertion of atransposable element or, at the least, such an insertion event cannot beruled out. In practice this may mean that a group of organisms aresegregated based on the presence (or absence) of the mutant phenotype.Those of skill in the art will understand that the pool of organisms tobe segregated should be grown or cultured under similar conditions toavoid segregation of phenotypes arising from non-genetic contributions(e.g., environmental effects). The method of the present invention canbe applied to any phenotype which can be distinguished and classified aseither wild-type or mutant. Such phenotypes can be detectable by visual,biochemical, agronomic, or morphological means. Those of skill willrecognise that the terms “wild-type” and “mutant” as used herein arearbitrary terms used to differentiate organisms according to thepresence or absence of a particular phenotype. The organisms to whichthe present invention can be applied can be prokaryotic or eukaryotic.Eukaryotic organisms can be haploid or diploid when employed in themethods of the present invention. In diploid organisms exhibiting thewild-type phenotype may be from the F1 generation, but mutant phenotypesassociated with transposon-tagged genes more commonly show up asrecessive mutants, and hence more commonly appear in the F2 generation.Thus, in a preferred embodiment of the invention, the organisms will befrom the F2 generation of a cross between a transposable element-donorindividual and a recipient inbred individual having no activetransposable elements. Preferably, the methods of the present inventionwill be applied to plants. In certain embodiments, the preferred plantis a monocot such as those of the family Gramineae, including suchexemplary species as Zea mays. In certain embodiments of the invention,the organisms having transposable elements will be maize plants from theF2 generation of a cross between a Mu-donor individual containing theMu-DR regulatory element (Chomet et al. (1991) Genetics 122:447457) andhigh copy number of Mu elements and a recipient inbred individual havingno active Mu elements. The genomic DNA of the organisms will have atleast one transposable element, and preferably a plurality oftransposable elements such as at least 5, 10, 25, 50, or 100.Transposable elements within the genome can be of the same or differingtypes. Organisms comprising transposable elements can be experimentallyderived according to methods available in the art. See, for example,Chomet (1994) in The Maize Handbook, ed. Freeling and Walbot(Springer-Verlag, New York), pp. 243-248. In a preferred embodiment, thetransposable element is Mutator (Mu). Robertson (1978) Mutation Res.51:21-28, Chandler and Hardeman (1992) Advances in Genetics 30:77-122).The terminal-inverted-repeat DNA (TIR) present in many transposableelements, including Mu, is well suited to the present invention.Insertion of a transposable element may occur within or near atransposable element-tagged gene's DNA sequence. The transposableelement-tagged gene to be identified with the method of the presentinvention may have a transposable element inserted within the gene'scoding sequence, such that transcription of the gene's normal functionalproduct is disrupted, leading to a mutant phenotype. Alternatively, thetagged gene may have a transposable element inserted within an intron,such that RNA splicing is affected, which in turn may disrupt thefunctional gene product, thereby yielding a mutant phenotype. Further,the tagged gene can have a transposable element inserted within a genecontrol region such as a promoter or enhancer element such that geneexpression is increased or decreased leading to a mutant phenotype. Foreach phenotype to which the method of the present invention is applied,at least one organism having a wild-type phenotype and at least onemutant are segregated. Optionally, at least 2, 4, 5, 10, 15, or 20organisms are present in the segregated wild-type population and atleast 2, 4, 5, 10, 15, or 20 are present in the segregated mutantpopulation.

The pooling of the isolated DNA can for instance be achieved using a3-dimensional pooling scheme (Vandenbussche et al, 2003, The Plant Cell,15, 2680-2693). The pooling is achieved preferably using equal amountsof DNA. The 3D-pooling scheme may comprise 10×10×10, resulting in 30pools (10+10+10) containing 10×10=100 different DNA samples per pool.Various other pooling strategies can be used with the present invention,examples thereof are multidimensional pooling (incl. 3-D pooling) orcolumn-, row- or plate pooling. In certain embodiments, pooling can alsobe performed before DNA extraction in the sampling stage, reducing thenumber of DNA preparations to 30 samples instead of 1000. (step (a) ofthe method).

The pooling step typically serves to identify the plant containing anobserved transposon insertion after one round of PCR screening. Poolingof the DNA further serves to normalise the DNAs prior to PCRamplification to provide for a more equal representation in thelibraries for sequencing. The DNA in the pools is restricted using atleast one restriction endonuclease. Depending on the case, i.e. size ofgenome or number of transposons, more endonucleases can be used. Incertain embodiments, 2 or more endonucleases can be used. For mostgenomes 2 endonucleases are sufficient and this is hence most preferred.In certain embodiments, especially for large or complex genomes, moreendonucleases can be used. Preferably the endonuclease provides forrelative short restriction fragments in the order of 50-500 bp, but thisis not essential. Typically, at least one frequent cutting endonucleaseis preferred, i.e. endonucleases that have a 4 or 5 base pairrecognition sequence. One such enzyme is MseI, but numerous others arecommercially available and can be used. Also enzymes that cut outsidetheir recognition sequence can be used (IIs type), or enzymes thatprovide blunt ended restriction fragments. A preferred combination usesone rare (6 and more base pair recognition sequence) and one frequentcutter.

After restriction of the pooled DNAs, or simultaneously therewith,adaptors are ligated to the restriction fragments to provide foradaptor-ligated restriction fragments. One or more different adaptorsmay be used, for instance two adaptors, one forward, one reverseadaptor. Alternatively one adaptor may be used for all fragments or setsof adaptors may be used that at the overhanging end of the adaptorcontain permutations of nucleotides such as to provide for indexinglinkers that may allow for a pre-selection step (Unrau et al., Gene,1994, 145, 163-169). Alternatively, blunt ended adaptors can be used, inthe case of blunt ended restriction fragments. Adaptor-ligation is wellknown in the art and is described inter alia in EP 534858. After adaptorligation, the pools of adaptor-ligated restriction fragments may be(pre-)amplified with a set of primers that are complementary to theadaptors. This may serve to (further) normalise the amount of DNA fromeach plant in the pools, or to increase the total amount of DNA in thepools to allow for multiple analysis of the pools (i.e. splitting upsamples) and to enhance the signal-to-noise ratio.

The adaptor-ligated restriction fragments are, after the optionalpre-amplification, amplified in step (d) of the method of the inventionwith a pair of primers. One of the primers is complementary to at leastpart of the adaptor and may further be complementary to part of theremainder of the recognition sequence of the endonuclease and mayfurther contain (randomly selected) selective nucleotides at its 3′-end,similar as is described in EP534858. The other primer in the set ofprimers is designed such that is capable of annealing to (part of) aborder of a transposon sequence. Typically, the primer overlaps with theconsensus sequence of the transposon, and preferably at the borderthereof. Preferably the primers are capable of selectively hybridisingunder stringent hybridisation conditions to the transposable element orthe adaptor, respectively. Alternatively, the primer may overlap (iscomplementary) with the transposon for at least 50, 60, 70, 80, 85, 90,95%. With an average length of a primer of about 20 bp, this amounts toan overlap of about 10 to 19 bases. This may be a consensus sequence oran actually known sequence of a transposon or transposon family in anorganism. Typical transposon sequences in plants are known, see forinstance: De Keukeleire et al. Chromosome Research, 2004, 12(2):117-123; Van den Broeck et al., The Plant Journal, 1998, 13(1), 121-129;Gerats et al, Plant Cell, 1990, 2, 1121-1128 describing the 284 bp dTph1transposition system in petunia. These references show that consensussequence are known for transposon families, in particular at the bordersof the transposons. Given these consensus sequence, design of suitableprimers can readily be achieved. For example, the Hat family (Hobo, Acand Tam3 in plants and animals. The transposon elements are known aswell as their sequence from the following articles: Atkinson P W, WarrenW D, O'Brochta D A (1993) The hobo transposable element of Drosophilacan be cross-mobilized in houseflies and excises like the Ac element ofmaize. Proc Natl Acad Sci USA 90: 9693-9697; Capy P, Vitalis R, LanginT, Higuet D, Bazin C (1996) Relationships between transposable elementsbased upon the integrase-transposase domains: is there a commonancestor? J Mol Evol 42: 359-368; Esposito T, Gianfrancesco F,Ciccodicola A et al. (1999) A novel pseudoautosomal human gene encodes aputative protein similar to Ac-like transposases. Hum Mol Genet. 8:61-67; Grappin P, Audeon C, Chupeau M C, Grandbastien M A (1996)Molecular and functional characterization of Slide, an Ac-likeautonomous transposable element from tobacco. Mol Gen Genet 252:386-397; Handler A M, Gomez S P (1996) The hobo transposable elementexcises and has related elements in tephridit species. Genetics 143:1339-1347; Hehl R, Nacken W K, Krause A, Saedler H, Sommer H (1991)Structural analysis of Tam3, a transposable element from Antirrhinummajus, reveals homologies to the Ac element from maize. Plant Mol Biol16: 369-371; Huttley G A, McRae A F, Clegg M T (1995) Molecularevolution of the Ac/Ds transposable element family in pearl millet andother grasses. Genetics 139: 1411-1419; Kempken F, Windhofer F (2001)The hAT family: a versatile transposon group common to plants, fungi,animals, and man. Chromosoma 110:1-9. Warren W D, Atkinson P W,O'Brochta D A (1995) The Australian bushfly Musca vetustissima containsa sequence related to transposons of the hobo, Ac and Tam3 family. Gene154: 133-134.

Preferably, the transposon directed primer is oriented and designed suchthat it faces outward of the targeted transposon. In certain embodimentto enhance the specificity, the one or both primers, preferably thetransposon directed primer, may contain nucleotides with improvedbinding affinity.

A part or segment of the adaptor-ligated restriction fragment isamplified using a pair of tagged primers, one or both of which may belabelled. Preferably, for each pool of each dimension, a differentprimer is used. In the above illustration this means that 30 forward anda single reverse primers are preferred. One of the forward and reverseprimer may be directed towards the adapter and the other of the reverseand forward primer may be directed to the targeted transposon.

Preferably each pair of primers (the adapter directed primer and thetransposon directed primer) may further comprise, dependently, one ormore of the following elements:

-   -   (i) a sequence primer binding site that can be used in the        following sequencing step,    -   (ii) a tag that serves to correlate the primer (and the        resulting amplification product) to the original member of the        population, and    -   (iii) a bead binding sequence that allows binding to the bead        that is used in the high throughput sequencing step.

In a typical embodiment the transposon directed primer can have thefollowing structure, both in 3′-5′ direction and in 5′-3′ direction:

Sequence primer binding site—optional Tag—Transposon specific PCR primersequence or Bead binding site—optional Tag—Transposon specific PCRprimer sequence.

In a typical embodiment the adapter directed primer can have thefollowing structure, both in 3′-5′ direction and in 5′-3′ direction:

Sequence primer binding site—optional Tag—adaptor specific PCR primersequence or Bead binding site—optional Tag—adaptor specific PCR primersequence.

In certain embodiments, both the transposon directed primer and theadapter directed primer can be provided with 1-10 randomly selectednucleotides at the 3′-end that, when used in amplification may providefor subsets. See FIG. 1. The length of the sequence primer binding siteand the transposon specific PCR primer sequence are those that areconventional in common PCR use, i.e., independently, from about 10 toabout 30 bp with a preference for from 15 to 25 bp. Preferably the partor segment of the adaptor ligated sequence that is amplified correspondsto a length that can be sequenced in one run using the high throughputsequencing technologies described below. In certain embodiments, thepart or segment has a length of between about 50 bp to about 500 bp,preferably from about 75 bp to about 300 bp and more preferably betweenabout 90 bp and about 250 bp. As stated above, this length may vary withthe sequencing technology employed including those yet to be developed.

Amplification with this set of primers will provide amplifiedadaptor-ligated restriction fragments (amplicons) of the flankingsequences of the targeted transposon in multiplex. By using primers(forward and/or reverse) containing a tag sequence that is unique foreach of the primers representing all pool dimensions, the specific poolorigin of each tag sequence is known as the sequence primer annealsupstream of the tag and as a consequence, the tag sequence is present ineach amplification product.

In certain embodiments, both forward and reverse primers are tagged. Inother embodiments, only one of the forward or reverse primers is tagged.The choice between one or two tags depends on the circumstances anddepends on the read length of the high throughput sequencing reactionand/or the necessity of independent validation. In the case of, e.g.,100 bp PCR products that are sequenced unidirectionally, only one tag isneeded. In the case of a 200 bp PCR product and a 100 bp read-length,double tagging is useful in combination with bi-directional sequencingas it improves efficiency 2-fold. It further provides the possibility ofindependent validation in the same step. When a 100 bp PCR product issequenced bi-directionally with two tagged primers, all traces,regardless of orientation, will provide information about the mutation.Hence both primers provide “address information” about which plantcontains which mutation. The tag can be any number of nucleotides, butpreferably contains 2, 3, 4 or 5 nucleotides. With 4 nucleotidespermuted, 256 tags are possible, whereas 3 nucleotides permuted provide64 different tags. In the illustration used, the tags preferably differby >1 base, so preferred tags are 4 bp in length. Amplification usingthese primers results in a library of tagged amplification products.

In certain embodiments, a system of tags can be used wherein theamplification process includes the use of a (1) a long primer comprising(a) a 5′-constant section linked to (b) a degenerate tag section (NNNN),linked to (c) a transposon or adaptor specific section-3′ and (2) ashort primer in subsequent amplifications that consists of (a) the5′-constant section linked to (b) non-degenerate tag section-3′ (i.e. aselection amongst NNNN). The long primer is preferably used in a shortmeasure and the short primer is used in an excess. The non-degeneratetag section can be unique for each pooled sample, for example, ACTG forpooled sample 1, AATC for pooled sample 2, etc. The short primer annealsto a subset of the long primer. The constant section of the primer canbe used as a sequence primer. The library preferably comprises equal,amounts of PCR products from all amplified pools. In the illustrativeexample, the library contains 1000 plants×100 bp=100 kb sequence to bedetermined for each transposon insertion site. In step (e) of themethod, the amplification products may be pooled, preferably in equal ornormalised amounts to thereby create a library of amplificationproducts. Exemplary, the complexity of the library will be 1000plants×250-500 bp=0.25-0.5 Mb sequence for each transposon insertionsite. The amplification products in the library may be randomlyfragmented prior to sequencing of the fragments. Fragmentation can beachieved by physical techniques, i.e. shearing, sonication or otherrandom fragmentation methods. In step (g), at least part, but preferablythe entire, nucleotide sequence of at least part of, but preferably ofall the fragments of step (d) or (f) is determined. In certainembodiments, the fragmentation step of the amplified products isoptional. For instance, when the read length of the sequencing techniqueand the PCR fragments length are about the same there is no need forfragmentation. Also in the case of larger PCR products, fragmentation ofthe amplified products may not be necessary if it is acceptable thatonly part of them are sequenced. For instance in case of 500 bp PCRproduct and read length of 100 (from each side) 300 bp remainunsequenced in case of no fragmentation prior to sequencing. The needfor fragmentation decreases with increasing read length of sequencingtechnology.

The sequencing may in principle be conducted by any means known in theart, such as the dideoxy chain termination method (Sanger sequencing).It is however preferred and more advantageous that the sequencing isperformed using high-throughput sequencing methods, such as the methodsdisclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005,WO 2004/070007, and WO 2005/003375 (all in the name of 454 LifeSciences), by Seo et al. (2004) Proc. Natl. Acad. Sci. USA 101:5488-93,and technologies of Helios, Solexa, US Genomics, etcetera, which areherein incorporated by reference. It is most preferred that sequencingis performed using the apparatus and/or method disclosed in WO03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007,and WO 2005/003375 (all in the name of 454 Life Sciences), which areherein incorporated by reference. The technology described currentlyallows sequencing of up to 40 million bases in a single run and is 100times faster and cheaper than competing technology. This will increasewith increasing read length per reaction and/or increasing numbers ofparallel reactions. The sequencing technology roughly consists of 5steps: 1) fragmentation of DNA and ligation of specific adaptor tocreate a library of single-stranded DNA (ssDNA); 2) annealing of ssDNAto beads, emulsification of the beads in water-in-oil microreactors andperforming emulsion PCR to amplify the individual ssDNA molecules onbeads; 3) selection of /enrichment for beads containing amplified ssDNAmolecules on their surface 4) deposition of DNA carrying beads in aPicoTiterPlate®; and 5) simultaneous sequencing in 100,000 wells bygeneration of a pyrophosphate light signal.

In a preferred embodiment, the sequencing comprises the steps of:

-   -   (1) annealing sequencing-adaptor-ligated fragments to beads,        each bead annealing with a single fragment;    -   (2) emulsifying the beads in water-in-oil micro reactors, each        water-in-oil micro reactor comprising a single bead;    -   (3) performing emulsion PCR to amplify adaptor-ligated fragments        on the surface of beads    -   (4) selecting/enriching beads containing amplified        adaptor-ligated fragments    -   (6) loading the beads in wells, each well comprising a single        bead; and    -   (7) generating a pyrophosphate signal.

In the first step (1), the adaptors that are present in the adaptorligated restriction fragments are annealed to the beads. As outlinedherein before, the sequencing adaptor includes at least a “key” regionfor annealing to a bead, a sequencing primer region and a PCR primerregion. In particular, the amplified adaptor-ligated restrictionfragments now contain at one of the ends the following sequence5′-Sequence primer binding site—Tag—Transposon specific PCR primersequence-3′, while at the other end a segment is present that may be asfollows: 5′-Bead annealing sequence—Tag—Adaptor specificsequence—restriction site specific sequence (optional)—(randomly)selective sequence (optional)-3′. It may be clear that the Sequenceprimer binding site and the Bead annealing sequence may be interchanged.This Bead annealing sequence can now be used for annealing the fragmentsto the bead, the bead carrying a nucleotide sequence to that end.

Thus, adapted fragments are annealed to beads, each bead annealing witha single adapted fragment. To the pool of adapted fragments, beads areadded in excess as to ensure annealing of one single adapted fragmentper bead for the majority of the beads (Poisson distribution).

In a preferred embodiment, to increase the efficiency of the transposonscreening further, it is beneficial to amplify the transposed-derivedPCR product directionally onto the bead for sequencing. This can beaccomplished to perform the transposon PCR with adaptor-tailed PCRprimers of which one strand of the adaptor on the MseI (or otherrestriction enzyme) side is complementary to the oligonucleotide coupledto the sequence beads. Hence the sequencing reaction will be primed fromthe transposon side (because sequencing occurs towards the bead),resulting in sequences that originate from the transposon outwards.

In a next step, the beads are emulsified in water-in-oil microreactors,each water-in-oil microreactor comprising a single bead. PCR reagentsare present in the water-in-oil microreactors allowing a PCR reaction totake place within the microreactors. Subsequently, the microreactors arebroken, and the beads comprising DNA (DNA positive beads) are enriched.

In a following step, the beads are loaded in wells, each well comprisinga single bead. The wells are preferably part of a PicoTiter™Plateallowing for simultaneous sequencing of a large amount of fragments.

After addition of enzyme-carrying beads, the sequence of the fragmentsis determined using pyrosequencing. In successive steps, thePicoTiter™Plate and the beads as well as the enzyme beads therein aresubjected to different deoxyribonucleotides in the presence ofconventional sequencing reagents, and upon incorporation of adeoxyribonucleotide a light signal is generated which is recorded.Incorporation of the correct nucleotide will generate a pyrosequencingsignal which can be detected. Pyrosequencing itself is known in the artand described inter alia on www.biotagebio.com;www.pyrosequencing.com/section technology. The technology is furtherapplied in e.g. WO 03/004690, WO 03/054142, WO 2004/069849, WO2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454Life Sciences), which are herein incorporated by reference. Aftersequencing, the sequences of the fragments that are directly obtainedfrom the sequencing step may be trimmed, preferably in silico, to removeany bead annealing sequence, sequencing primer, adaptor or transposonrelated sequence information. This may result in a better alignment inthe next step with known sequences from the database to identify anypossible hits. By doing this in silico, the information provided by thetag may be preserved in a separate database field so as to later onconnect the discovered mutated gene to the address in the DNA pools.

Typically, the alignment or clustering is performed on sequence datathat have been trimmed for any added adaptors/primer and/or identifiersequences i.e. using only the sequence data from the fragments thatoriginate from the nucleic acid sample.

Methods of alignment of sequences for comparison purposes are well knownin the art. Various programs and alignment algorithms are described in:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-244; Higgins andSharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucl. Acids Res.16:10881-90; Huang et al. (1992) Computer Appl. in the Biosci. 8:155-65;and Pearson et al. (1994) Meth. Mol. Biol. 24:307-31, which are hereinincorporated by reference Altschul et al. (1994) Nature Genet. 6:119-29(herein incorporated by reference) present a detailed consideration ofsequence alignment methods and homology calculations. The NCBI BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1990) is availablefrom several sources, including the National Center for BiologicalInformation (NCBI, Bethesda, Md.) and on the Internet, for use inconnection with the sequence analysis programs blastp, blastn, blastx,tblastn and tblastx. It can be accessed at<http://www.ncbi.nlm.nih.gov/BLAST/>. A description of how to determinesequence identity using this program is available at<http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html>. The databasepreferably comprises EST sequences, genomic sequences of the species ofinterest and/or the non-redundant sequence database of GenBank orsimilar sequence databases. High throughput sequencing methods can beused as described in Shendure et al. Science, Vol 309, Issue 5741,1728-1732. Examples thereof are microelectrophoretic sequencing,Hybridization sequencing/sequencing by hybridization (SBH), cyclic-arraysequencing on amplified molecules, cyclic-array sequencing on singlemolecules, , Non-cyclical, single-molecule, real-time methods, such as ,polymerase sequencing, exonuclease sequencing, nanopore sequencing.

For an optimal result, it is of interest that the fragments or theamplified products are sequenced with sufficient redundancy. Redundancyis what enables making the distinction between sequencing errors andgenuine genome sequences. In certain embodiments, the redundancy of thesequencing is preferable at least 4, more preferably at least 5, but ascan be seen from the illustration, redundancies of more than 6,preferably more than 8 or even more than 10 are considered advantageous,although not essential for the inventive concept.

In step (i) of the method, the fragments are identified that yield a hitin the database and that hence may be linked to a gene or a phenotype ofinterest. Based on this information, the tags can be used to identifythe pool and/or the plant. Based on the hit in the database, a probe canbe designed that allows for the identification of the gene of interest.

DESCRIPTION OF THE FIGURES

FIG. 1: In the distribution analysis of dtph1 transposon flankingsequences., the general built-up of a sequence tag, as consisting of(from right to left) a unique genomic sequence, the transposon (invertedrepeat) sequence and the 3D tag is described. The population of 100plants is organised according to a 3D grid (10*10*10) in which eachplant is identified according to a unique 3D coordinate (x,y,z)reflecting its position along the x, y and z axis. Dimensions X1 to X10correspond to sequence tag numbers 1 to 10, similar for Y and Z. The tagcodes in the figures are translated into tag # in the sequencename ,e.g. AGAC corresponds to tag07. The picture shows the 3D hit in plantwith pool coordinates (3, 17, 24)

FIG. 2 describes the result of a blast search with a specific genicsequence, the Petunia transcription factor NAM-like 3 gene (no apicalmeristems like, gj|21105733|gb|AF509866.1), against the insertionflanking sequences database; an insertion hit is identified with thecoordinates 2, 12, 30. This result demonstrates that insertions inspecific homologous coding sequences can be traced.

FIG. 3 describes the result of a blast search with a specific butheterologous genic sequence, an Arabidopsis AGL62 MADS box gene, againstthe database; an insertion hit is identified in plant 9, 17, 29; thishit specifies a hitherto unknown potential MADS box gene in Petunia andits corresponding mutant. This result demonstrates that insertions inspecific heterologous coding sequences are traced successfully.

FIG. 4 provides a sequence analysis wherein a subset of 230.000 of theavailable 318.000 sequences has been completely ordered according tothree levels:

-   -   1) Sequence identity of the flanking sequences (ordering        according to insertion site). All sequences identifying the same        insertion are called one group.    -   2) Within groups, according to their differing 3D sequence tags.    -   3) According to copy number of sequences belonging to a group.

The following graphs have been extrapolated based on the analysis of 20%from 230.000 ordered sequences (of a total of 318.000 sequences). Tofacilitate interpretation of these graphs, 3 groups of sequences areshown in this figure, representing 3 independent transposon insertionsites. The first example identifies four sequences, with the respective3D tags spanning positions 5-8, followed by the Inverted Repeat sequenceof the transposon, ending at position 22, followed by a stretch ofgenomic sequence. The coordinates 6-20-29 define this sequence asbelonging to the plant at that particular coordinate of the population.Tag01 to tag10: X dimension, Tag11 to tag20: Y dimension, Tag21 totag30: Z dimension.

FIG. 5 provides a graphic display of the relative dimensionaldistribution versus copy number occurrence.

FIG. 6: Of the 3500 sequence tags that had three copies, 294 had 3unique coordinates, meaning that these could trace back these sequencesto their plant of origin. For the other copy classes those numbers were532 for the four-copy class; 622 for the five-copy class; 478 for thesix-copy class; and 1500 for the remaining classes. This implies that intotal over 3000 sequence tags have been identified (out of 230.000 ofthe 318.000 available) that could be related back to their plant oforigin.

FIG. 7: 4 copy number classes and their relative contribution to totalestimated 3D hit number and total number of sequences in 3D 454transposon library

FIG. 8: Number of # insertion sites (groups) versus copy number (fullrange) An analysis of the number of copies per sequence tag showed thatamongst the 230.000 of the analysed subset, there were nearly 16.000unique fragments; 7500 fragments had two copies; 3500 had three copies;2500 had four copies; 1500 had five copies; 1000 had six copies; 1350had 7 or 8 copies; 1100 had 9-11 copies; 1400 had 12-20 copies; 950 had21-40 copies; while the remainder had the remaining copies.

FIG. 9: provides a graphic display of some results. Subset of 253.394sequences analyzed (total 318.000), Only 1% of the sequences did notcontain a recognizable Tag (depicted as ??, right column) An analysis of20% of the subset of 230.000 sequence tags indicated a good distributionof sequence tags over the different pooled samples of the population andranged from over 6000 for coordinate 23 to nearly 30.000 for coordinate15; the average being around 8500. Less then 1% of the fragments couldnot be assigned to a specific coordinate.

FIG. 10: Schematic illustration of a transposon targeted in a MseI-ECORIrestriction fragment using an adaptor directed primer and a transposondirected primer carrying a tag and a bead annealing sequence.

FIG. 11: Schematic representation of an amplified adaptor-ligatedfragment annealed to a bead via a bead annealing sequence (B). Thefragment contains tags (T1 and/or T2), the adaptor (AD), eventualremains of the restriction site (RE), the sequence of the fragmentitself (SEQ), the transposon specific primer sequence (TR) and thesequence primer binding site (SPBS) used for the initiation of thesequencing step.

EXAMPLES

The present invention is illustrated with the following examples thatprovide illustration of the principle. Screening a transposon populationis advanced by using novel high-throughput sequencing methods, such asthat of 454 Life Sciences. With the current state-of-the-art, 454 LifeSciences technology produces approximately up to 40 Mb sequence in asingle sequencing run. A limitation at present is that read lengths areapproximately 100-200 bp/read. Assuming the screening of a populationconsisting of 3072 plants harbouring on average 200 transposons toidentify transposon tagging of a particular gene, the approach is asfollows:

1) Genomic DNA of 3072 plants of the transposon population is isolated;2) A 3-dimensional pooling scheme of equal amounts of DNA per plant isset up (e.g. 15×15×14), resulting in 44 pools (15+15+14=44) containing3072/14=219 or 3072/15=205 different DNA samples (Vandenbussche et al.,2003);

This pooling step serves to be able to identify the individual plantcontaining an insertion directly from the sequence data. Pooling ofgenomic DNAs further serves to normalise DNAs prior to PCR amplificationto increase chances that all DNAs are represented equally in thesequence library;

3) adaptor ligated restriction fragment templates (AFLP templates, seeEP534858, Vos et al NAR 1995, 23, 4407) are prepared from all 44 pooledDNAs using a single restriction enzyme that cuts the genome every250-500 bp (e.g. using a 4- or 5 cutter; e.g. MseI);4) unidirectional PCR amplification is carried out using a PCR primerlocated at the border of the transposon sequence and facing outwards anda non-selective adaptor primer, to amplify the flanking sequences of alltransposon in multiplex. Per plant containing 200 transposons, thisyields 200× approximately 250 bp=50 kb flanking sequence per borderside, of which 20 kb will be sequenced in case of 100 bp read lengths.For 3072 plants this equals 153 Mb flanking sequence of which 61 Mb issequencable in case of 100 bp sequence read lengths;5) Equal amounts of PCR products from all 44 wells are pooled to createa pooled PCR product library;6) The pooled PCR product library is sequenced using 454 Life Sciencessequencing-by-synthesis technology without further fractionation of PCRproducts. Output is approximately 200,000 100 bp sequences, representing0.33×(20/61 Mb) coverage on average of all flanking sequences of 3072plants. Hence at least 3 sequence runs are needed in order to target thevast majority of all flanking sequences of all 3072 plants;7) resulting sequences are Blasted to identify hits with ESTs or genomesequences;8) plants carrying transposon insertions in genes of interest areidentified based on their tags and optionally probes or PCR primers aregenerated to confirm this.

Example 1

A population of 1000 Petunia W138 plants was sampled according to the3-Dimensional strategy as described by Vandenbussche et al. (2003) andothers, resulting in 30 pooled samples (X1-X10, Y1-Y10 and Z1-Z10),covering every individual of the whole population with threecoordinates. This enabled tracing back the origin of any specific PCRproduct to the plant of origin within the population.

The DNA samples were then digested with an enzyme, cutting inside thetransposon and an enzyme cutting at a specific but random position inthe flanking genomic DNA.

Adapters were then ligated to allow subsequent PCR amplification of alldigested fragments. A biotinylated adapter was ligated to the internaltransposon site. The DNA samples were then purified and biotinylatedfragments collected by adding Streptavidin beads and using a magnet. Allflanking sequences from all transposon insertions present in every DNApool were then amplified using an adapted transposon display protocol(VandenBroeck et al., 1998). For every pooled sample, X1-X10, Y1-Y10 andZ1-Z10, in the example, a different transposon primer was used,incorporating the corresponding pool coordinate as a 4 nucleotide codein its 5′ end (3D-tag).

All PCR products were subsequently pooled in three superpools, one foreach dimension, to enable normalization of the samples, according toprocedures described in the art; with this step, fragments that arepresent in every individual and thus in every sample, are diminished inoccurrence. This prevents over-representation of fragments in thesamples-to-be-sequenced.

The obtained collections of single-stranded molecules were converted todouble-stranded molecules by a single round of PCR amplification with aspecific primer, harboring a MunI site.

The obtained products were digested with MunI/MseI in order to enablethe subsequent ligation of adapter sequences that allow either furtheramplification or direct 454(G20) sequencing.

The three samples then were pooled in one superpool and subjected to theRoche GS20/454 sequencing procedure as described by the manufacturer.

A protocol was developed for the amplification of transposon flankingsequences by Transposon Display and subsequent high throughputsequencing from a population of 1000 plants.

Overview of the Procedure

An overview of the procedure is given below:

-   -   DNA preparation (1000 plants sampled in 3D fashion, resulting in        30 pooled DNA's)    -   MunI/MseI digest (ca 5 μg of Pooled DNA)    -   Bio-Mun & Mse adaptor ligation    -   Purification (PCR purification columns, to get rid of bio-Mun        adaptors and very small fragments)    -   Beads extraction (enrichment of Mun/Mse fragments)    -   Transposon Display PCR amplifications:    -   Pre-amp with MunACAC & Mse+0 primers (enrichment of Transposon        flanking sequences)    -   Selective PCR with Pooled specific IR**outw & Mse+0 primers        (Amplification of Transposon Flanking Sequences)    -   Second pooling into “Block”, “Row” and “Column” pools    -   Normalization    -   Conversion to double-stranded molecules    -   MunI/MseI digestion    -   454-Mun-B & 454-Mse-A adaptor ligation    -   PCR amplification with bio-AmpB & AmpA primers    -   Final pooling into one sample    -   454 sequencing

DNA Preparation

1000 plants sampled in 3D fashion, resulting in 30 DNA samplesrepresenting 100 plants each; procedure according to Vandenbussche etal., Plant Cell 15 (11): 2680-2693 (2003)

Munh/MseI digestion (ca. 5 μg)30 samples

Ca. 5 μg DNA in 50 μl H₂O

Add 20 μl mix:   2 μl MunI (10 U/μl stock)   2 μl MseI (10 U/μl stock)  7 μl NEB 4 (10 × stock) 0.7 μl BSA (100 × stock) H₂O to 20 μl

Incubate: 1.5 hrs. at 37° C.

Adapter ligation:

Add 30 μl mix   8 μl MunI-bio-Adapter (5 pmol/μl stock)   8 μlMseI-Adapter (50 pmol/μl stock)   3 μl NEB 4 (10 × stock) 0.3 μl BSA(100 × stock)   3 μl ATP (10 mM stock)   3 μl T4 DNA ligase (5WeissU/μlstock) H₂O to 30 μl

Incubate: 4 hrs. at 37° C.

Adapter Sequences:

Mun I (blo) adapter: bio-5′-CTCGTAGACTGCGTACG-3′ 3′-CTGACGCATGCTTAA-5′Mse I adapter: 5′-GACGATGAGTCCTGAG-3′ 3′-TACTCAGGACTCAT-5′

Purification 30 Samples

Purify the DNA's, using the Qiagen PCR purification kit Elute with 55 μlEB buffer (5 μl on 1.5% agarose gel)

Beads Extraction 30 Samples

Wash 25 μl streptavidine beads (ca. 0.1 mg MyOne beads, streptavidin C1)once in 200 μl STEX, and resuspend in 100 μl binding buffer.

STEX: Binding buffer: 10 mM Tris.Cl (pH 8.0) 10 mM Tris.Cl (pH 8.0) 1 MNaCl 2 M NaCl 1 mM EDTA 1 mM EDTA 0.1% Triton X-100 0.1% Triton X-100

Add 100 μl diluted (&washed) Streptavidine beads to the 500 μlrestriction/ligation mixture and incubate for 60 minutes on a rotator,at room temperature. Collect the beads, using the magnet and remove thesupernatant. Wash the beads with 200 μl STEX and transfer to anothertube. Wash the beads three times with 200 μl STEX and resuspend thebeads finally in 50 μl T₀₁E, transfer to another tube (remove the STEXwell).

-   -   T₀₁E:    -   10 mM Tris.Cl (pH8.0)    -   0.1 mM EDTA

Transposon Display PCR Amplifications: Pre-Amplification 30 Samples

Take 2 μl template DNA (mix the beads well, the DNA fragments are stillconnected) and add:

18 μl mix: 0.6 μl Mun + ACAC primer (10 μM) 0.6 μl Mse + 0 primer (10μM) 0.8 μl dNTP (5 mM)   2 μl 10 × PCR buffer   2 μl MgCl₂ (25 mM) 0.6 URed Hot Tag DNA polymerase H₂O to 18 μland incubate them according to the following PCR profile (PE 9600):

30″ 94° C. 15″ 94° C. | Touchdown: 30″ 65° C. >> 56° C. ({circumflexover ( )}t = −0.7° C./cycle) |13 cycles 60″ 72° C. | 15″ 94° C. | 30″56° C. | 22 cycles 60″ 72° C. |

Primer Sequences:

Mun I + ACAC: 5′-AGACTGTGTACGAATTGACAC-3′ Mse I + 0:5′-GACGATGAGTCCTGAGTAA-3′

Analyse 5 μl on 1.5% agarose gel, and dilute the samples 10 times withH₂O and perform a selective PCR amplification:

Transposon Display PCR Amplifications:

Take 5 μtemplate DNA and add:

45 μl mix: 1.5 μl IR_(outw) primer (10 μM)* 1.5 μl Mse + 0 primer (10μM)   2 μl dNTP (5 mM)   5 μl 10 × PCR buffer   5 μl MgCl₂ (25 mM)   1 URed Hot Taq DNA polymerase H₂O to 45 μl

And incubate them according to the following PCR profile (PE 9600).

30″ 94° C. 15″ 94° C. | Touchdown: 30″ 65° C. >> 56° C. ({circumflexover ( )}t = −0.7° C./cycle) |13 cycles 60″ 72° C. | 15″ 94° C. | 30″56° C. | 22 cycles 60″ 72° C. |

Primer Sequences:

IR_(outw): * 5′-CATATATTAANNNNGTAGCTCCGCCCCTG-3′every pooled sample is amplified with a unique IR_(outw) primer,specified by the NNNN positions; this allows to allocate obtainedsequences to their co-ordinate of origin.

Mse I + 0: 5′-GACGATGAGTCCTGAGTAA-3′Second Pooling 30 Samples into 3 Samples

Pool the PCR products from the ten samples from each dimension to create3 samples: column/row/block

Normalization.

In order to enhance the amount of unique fragments against a backdrop offragments shared by many or all individuals, the second pooled samplesare normalized, based on conventionally known procedures. The procedureinvolves a hybridisation and a purification step for obtaining singlestranded molecules.

Hybridisation (about: 10 μg Each Sample) 3 Samples

Precipitate the DNA's of the Pooled samples and dissolve in 15-35 μl

Add (relative volumes) to 15 μl formamid: 4.5 μl TE   3 μl H₂OHeat to 80° C. under mineral oil for 3 minutes

Add   3 μl bufferA 4.5 μl H₂OIncubate the probe O/N at 30° C.

-   -   Buffer A:    -   0.1 M Tris.Cl (pH8.0)    -   1.2 M NaCl    -   50 mM EDTA

Purification by HAP Chromatography 3 Samples

Single stranded molecules are selected for by standard HAPchromatography as described by de Fatima Bonaldo et al., GenomeResearch, 6: 791-806 (1996) and subsequently converted to double-strandmolecules.

Conversion to Double-Stranded Molecules 3 Samples

One PCR cycle with “Mse+0 with Mun site” primer add to 50 μl sample:

25 μl mix: 5 μl MIBUS 796 (10 μM) 4 μl dNTP (5 mM) 7.5 μl 10 × PCRbuffer 2.5 μl MgCl₂ (50 mM) 0.2 μl PlatinumTaq DNA polymerase H₂O to 25μlPrimer sequence:

MIBUS 796: 5′-CATATACAATTGGACGATGAGTCCTGAGTAA-3′

And incubate them according to the following profile (PE 9600):

 2′ 94° C.  1′ 56° C. 10′ 72° C.Munh/Mse digest 3 samples

Template DNA in 65 μH₂O

Add 25 μl mix:   2 μl MunI (10 U/μl stock)   2 μl MseI (10 U/μl stock)  9 μl NEB 4 (10 × stock) 0.9 μl BSA (100 × stock) H₂O to 25 μl

Incubate: 1.5 hr. at 37° C.

454 Adapter ligation

Add   4 μl MunI-bio-Adapter B (50 pmol/μl stock)   4 μl MseI-Adapter A(50 pmol/μl stock)   2 μl NEB 4 (10 × stock) 0.2 μl BSA (100 × stock)  3 μl ATP (10 mM stock)   3 μl T4 DNA ligase (5WeissU/μl stock) H₂O to20 μl

Incubate: 4 hrs. at 37° C.

Adapter sequences:

Mun I adapter B: MIBUS 8035′-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAG-3′ MIBUS7953′-AGGGGACACACGGAACGGATAGGGGACAACGCACAGAGTCTTAA-5′ Mse I adapter A:MIBUS 800 5′-CCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAG-3′ MIBUS 8013′-GAGTAGGGACGCACAGGGTAGACAAGGGAGGGACAGAGTCAT-5′PCR-aanplification for 454 sequencing 3 samples

Amplification adaptor primer A & B: MIBUS 803bio-5′-CCTATCCCCTGTGTGCCTTG-3′ MIBUS 802        5′-CCATCTCATCCCTGCGTGTC-3′Final Pooling 3 Samples into 1 Sample

Pool the samples to create 1 superPool, ready for High throughputsequencing

454 Sequencing 1 Sample

pGEM-T Cloning for Insert Size Distribution Test1 Sample

In order to test the efficiency of the normalisation procedure, werandomly isolated 22 fragments in order to determine their sizedistribution. Take 1 μl PCR mix (from the superpool sample for 454sequencing)

Add 4 μl mix: 1 μl pGEM-T (4 times diluted) 2.5 μl 2 x rapid ligationbuffer 0.25 μl Ligase H₂O to 4 μl

Incubate: 3 hr. at 37° C.

Transform into E. coli (DH5α cells)Plate 100 μl onto LB⁻ Amp plates

Incubate: o/n at 37° C.

Pick 22 coloniesPerform a PCR on boiled preps.with the AmpA/AmpB primersAnd run on a 2% agarose gel:

Results:

A database of 318.000 sequence tags of on average 102 basepairs wasobtained. A subset of 230.000 sequences has been completely orderedaccording to three levels:

1) Sequence identity of the sequence, flanking the inverted repeat ofthe transposon (ending with CCGCCCCTG). All sequences identifying thesame insertion are called one group.2) Within each group, sequences are ordered according to the different3D tags in their 5′ sequence.3) According to copy number of sequences belonging to a group.

The data have been extrapolated based on the analysis of 20% from230.000 ordered sequences (of a total of 318.000 sequences). Theanalysis is described in the FIGS. 1-9.

TABLE SEQ ID sequence # Mun I (bio) bio-5′-CTCGTAGACTGCGTACG-3′ 1adapter 3′-CTGACGCATGCTTAA-5′ 2 Mse I adapter 5′-GACGATGAGTCCTGAG-3′ 33′-TACTCAGGACTCAT-5′ 4 Primer sequences Mun I + ACAC:5′-AGACTGTGTACGAATTGACAC-3′ 5 Mse I + 0: 5′-GACGATGAGTCCTGAGTAA-3′ 6IR_(outw): * 5′-CATATATTAANNNNGTAGCTCCGCCCCT 7 G-3′ Mse I + 0:5′-GACGATGAGTCCTGAGTAA-3′ 8 MIBUS 796: 5′-CATATACAATTGGACGATGAGTCCTGAG 9TAA-3′ Adapter sequences Mun I adapter 5′-CCTATCCCCTGTGTGCCTTGCCTATCCC10 B: MIBUS 803 CTGTTGCGTGTCTCAG-3′ MIBUS7953′-AGGGGACACACGGAACGGATAGGGGACA 11 ACGCACAGAGTCTTAA -5′ Mse I adapter5′-CCATCTCATCCCTGCGTGTCCCATCTGT 12 A:MTBUS 800 TCCCTCCCTGTCTCAG-3′ MIBUS801 3′-GAGTAGGGACGCACAGGGTAGACAAGGG 13 AGGGACAGAGTCAT-5′ Amplificationadaptor primer A & B: MIBUS 803 bio-5′- CCTATCCCCTGTGTGCCTTG -3′ 14MIBUS 802 5′- CCATCTCATCCCTGCGTGTC -3′ 15

1. Method for the identification of an insertion associated with a geneor sequence of interest in a member of a transposon population,comprising the steps of: (a) Isolating, individually or in pools,genomic DNA of the transposon population; (b) optionally, pooling theDNA obtained in step (a); (c) restrict the DNA using one or more,preferably two or more, most preferably two, restriction endonucleases,preferably at least one of which is a frequent cutting restrictionendonuclease that does not cut in the transposon and preferably at leastone is a rare cutting restriction endonuclease that cuts in thetransposon, ligate adaptors to the restriction fragments to therebyprepare adaptor-ligated restriction fragments; (d) amplifying theadaptor-ligated restriction fragments with a pair of (optionallylabelled) primers, whereby one of the primers comprises a section thatis complementary (capable of hybridising) to part of a (known)transposon sequence and further contains a sequence primer binding site,wherein the other primer is at least complementary to the adaptor,wherein one or both primers contain a tag; (e) optionally, pooling theamplification products of step (d) to create a library of amplificationproducts; (f) optionally, fragmenting the amplification products in thelibrary; (g) determining the nucleotide sequence of the fragments of(d), (e) or (f) using high throughput sequencing; (h) optionally,trimming the sequence of the fragments in silico to thereby remove anyadaptor and/or transposon related sequence information; (i) identifyingone or more fragments of step (g) or (h) that are capable of aligningwith nucleotide sequences from a database, thereby correlating thenucleotide sequences from the database with the phenotype of interest;(j) identifying the member(s) of the transposon population containingthe fragment(s) of step (i); (k) optionally, designing a probe or PCRprimer pair based on the fragments of step (i) and using it to confirmtransposon insertion in the gene of interest in the genome of the memberidentified in (I).
 2. Method according to claim 1, wherein the poolingis a 3D-pooling strategy.
 3. Method according to claim 1 wherein thedatabase comprises EST sequences, genomic sequences of the species ofinterest and/or the non-redundant sequence database of GenBank orsimilar sequence databases.
 4. Method according to claim 1, wherein thehigh throughput sequencing is based on Sanger sequencing, preferably bycapillary electrophoresis.
 5. Method according to claim 1, wherein thehigh throughput sequencing is sequencing-by-synthesis, preferablypyrosequencing.
 6. Method according to claim 1, wherein sequencing isperformed on a solid support such as a bead.
 7. Method according toclaim 6, wherein sequencing comprises the steps of: (1) annealingsequencing-adaptor-ligated fragments to beads, each bead annealing witha single fragment; (2) emulsifying the beads in water-in-oil microreactors, each water-in-oil micro reactor comprising a single bead; (3)performing emulsion PCR to amplify adaptor-ligated fragments on thesurface of beads (4) selecting/enriching beads containing amplifiedadaptor-ligated fragments (6) loading the beads in wells, each wellcomprising a single bead; and (7) generating a pyrophosphate signal. 8.Method according to claim 1, wherein at least one of the primerscontains one or more nucleotides with improved binding affinity.